Klaus Gerwert

Electrostatic calculations of the pKa values of ionizable groups in bacteriorhodopsin

The effects of solvation and charge-charge interactions on the pKa of ionizable groups in bacteriorhodopsin have been studied using a macroscopic dielectric model with atom-level detail. The calculations are based on the atomic model for bacteriorhodopsin recently proposed by Henderson et al. Even if the structural data are not resolved at the atomic level, such calculations can indicate the quality of the model, outline some general aspects of electrostatic interactions in membrane proteins, and predict some features. The effects of structural uncertainties on the calculations have been investigated by conformational sampling. The results are in reasonable agreement with experimental measurements of several unusually large pKa shifts (e.g. the experimental findings that Asp96 and Asp115 are protonated in the ground state over a wide pH range). In general, we find that the large unfavorable desolvation energies of forming charges in the protein interior must be compensated by strong favorable charge-charge interactions, with the result that the titrations of many ionizable groups are strongly coupled to each other. We find several instances of complex titration behavior due to strong electrostatic interactions between titrating sites, and suggest that such behavior may be common in proton transfer systems. We also propose that they can help to resolve structural ambiguities in the currently available density map. In particular, we find better agreement between theory and experiment when a structural ambiguity in the position of the Arg82 side-chain is resolved in favor of a position near the Schiff base.

A model-independent approach to assigning bacteriorhodopsin's intramolecular reactions to photocycle intermediates

By using factor analysis and decomposition, bacteriorhodopsins intramolecular reactions have been assigned to photocycle intermediates. Independent of specific kinetic models, the pure BR-L, BR-M, BR-N, and BR-O difference spectra were calculated by analyzing simultaneously two different measurements in the visible and infrared spectral region performed at pH 6.5, 298 K, 1 M KCl, and pH 7.5, 288 K, 1 M KCl. Even though after M formation L, M, N, and O intermediates kinetically overlap under physiological conditions, their pure spectra have been separated by this analysis in contrast to other approaches at which unphysiological conditions or mutants have been used or specific photocycle models have been assumed. The results now provide a set reference spectra for further studies. The following conclusions for physiologically relevant reactions are drawn: (a) the catalytic proton release binding site, asp 85, is protonated in the L to M transition and remains protonated in the intermediates N and O; (b) the catalytic proton uptake binding site asp 96 is deprotonated in the M to N transition and already reprotonated in the N to O transition; (c) proton transfer between asp 96 and the Schiff base is facilitated by backbone movements of a few peptide carbonyl groups in the M to N transition.

Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy

Bacteriorhodopsins proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 mus, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k(7) = 0.3 ms, k(4) = 2.3 ms, k(3) = 6.9 ms, k(6) = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k(4)), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M(1) to M(2) transition. However, experimentally two different M intermediates are not resolved, and M(2) and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M(2)) transition. This reaction is proposed to be involved in the reset switch of the pump, (b) In the M to N (conceptionally M(2)) transition, most likely, asp-85s carbonyl frequency shifts from 1,762 to 1,753 cm(-1) and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.

Proline residues undergo structural changes during proton pumping in bacteriorhodopsin

The role of proline residues in the proton pump mechanism of bacteriorhodopsin is investigated by Fourier‐transform infrared (FTIR) difference spectroscopy using [15N]‐proline labelled bacteriorhodopsin. Due to the isotopic shifts of absorbance bands, proline vibrations can be assigned to bands at 1616 cm−1 and between 1460 cm−1 and 1400 cm−1. At 1616 cm−1 a decrease of an absorbance band is observed in the intermediates K, L, M and in the dark adapted state (DA). This absorbance change can be correlated to a light induced H‐bond alteration of a X‐Pro amide carbonyl group. Isotopic shifts of difference bands between 1450 cm−1 and 1400 cm−1 are detected in K and M but are not observed in L and DA. From these frequency shifts of proline vibrations it is concluded that two proline residues undergo structural changes of their X‐Pro C‐N peptide bonds during proton pumping. The results support the suggestion that the more flexible X‐Pro C‐N bonds are used as hinges for a functional important structural motion of the protein backbone.

Only water-exposed carboxyl groups are protonated during the transition to the cation-free blue bacteriorhodopsin

FTIR (Fourier‐transform‐infrared) difference spectra between cation‐free blue and purple bacteriorhodopsin were recorded. The results indicate that during the blue to purple transition no isomerization of the chromophore takes place. It is further observed that approx. 14 water‐exposed carboxyl groups ofamino acids are protonated in blue bacteriorhodopsin. The groups were deprotonated during the blue to purple transition. Protonation of carboxyl groups in the interior of the protein, which are not accessible to water, can be excluded.

FTIR studies on crystals of photosynthetic reaction centers

It is shown that high‐quality light‐induced FTIR‐difference spectra can be obtained from reaction center crystals of Rhodopsendomonas viridis. Difference spectra between the ground state, PQ, and a light‐activated state, P+Q−, have been recorded. The difference spectra are in good agreement with those reported previously for reaction centers reconstituted into lipid vesicles [(1985) FEBS Lett. 187, 227–232]. This good correspondence indicates that in both sample preparations the same intramolecular processes take place during this transition. In addition to measurements of absorbance changes in the visible spectral region, which indicate reactions of the chromophores and their microenvironments, those in the infrared spectral region also show that the protein side groups and backbone undergo the same light‐induced changes in the crystals. It is observed that, besides the porphyrin ring system, the C9 = O keto and ester groups of, most likely, the primary donor, undergo light‐induced changes in charge distribution during oxidation of the primary donor. Large conformational changes of the protein backbone can be excluded for the observed transition.

Investigation of Quinone Reduction in Rhodopseudomonas viridis by FTIR Difference Spectroscopy and X-Ray Diffraction Analysis

The crystallization [1] and subsequent x-ray diffraction analysis [2,3] of the reaction center from Rps. viridis have provided a detailed picture of the chromophores in their ground states, PIQAQB. The crystals exhibit linear dichroism [4,5] and have been shown to be photoactive [4]. A series of experiments was therefore undertaken to investigate various charge-separated states stabilized by continuous illumination: (1) difference Fourier analysis of x-ray diffraction data sets for illuminated and dark crystals was performed for reaction centers reconstituted with ubiquinone-9, yielding differences between P+QAQB − and PQAQB, and (2) light-induced FTIR difference spectra were measured for crystals and for reaction centers reconstituted into lipid vesicles to identify differences between Q A − and Q B − . Furthermore, using reconstituted reaction center samples it was possible to selectively stabilize P+QA −, P+QB −, PI−, PI−QA − and PI−QA 2−. Thus the electron transfer pathway could be followed from the primary donor, P, to the secondary acceptor, QB, via the intermediary (bacteriopheophytin) acceptor, I, and QA.

FTIR Studies of Light-Induced Intramolecular Processes on Crystals and Reconstituted Reaction Centers from Rhodopseudomonas viridis

The crystallization (1) and subsequent x-ray diffraction analysis (2,3) of the reaction center from Rps. viridis have provided a detailed picture of the ground state, PIQ A Q B . In order to gain insight into the electron transfer dynamics of the protein-chromophore complex, light-induced FTIR difference spectra have been measured on RC crystals and on RC’s reconstituted into lipid vesicles. In contrast to Resonance Raman spectroscopy which yields information only on the conjugated parts of chromophores, infrared spectroscopy is able to monitor absorbance bands of all molecular groups in the protein-pigment complex. FTIR difference spectra of the various charge-separated states in the reaction center should additionally provide information on non-chromophoric groups in the protein and thereby a better understanding of the molecular events in the primary electron transfer reactions of photosynthesis.

Investigation of intramolecular processes in proteins by time resolved FTIR-difference spectroscopy

A new technique is developed by which intramolecular processes of proteins can be investigated time-resolved in the infrared spectral region. By this technique the transduction mechanism of light energy into chemical energy in chromoproteins is explored on an atomic resolution.

Time-resolved FTIR studies on proteins

Two different time-resolved FTIR techniques are used to investigate the light-driven proton pump bacteriorhodopsin: (1) the rapid scan technique yielding a few ms time-resolution, and (2) the stroboscopic technique yielding a few microsecond(s) time-resolution.